1. Field of the Invention
The present invention relates to a membrane for Western blotting which has a three-dimensional open pore structure, an average pore diameter of 0.1-1.0 μm and a thickness of 30-200 μm, wherein the membrane for Western blotting is manufactured by subjecting nanofibers having an average fiber diameter of 50-1000 nm, obtained by electrospinning, to a hot-plate calendering process, and a method for manufacturing the same.
2. Description of the Prior Art
Membranes for Western blotting which are currently commonly used include porous membranes made of nitrocellulose (NC), nylon or polyvinylidene fluoride (hereinafter referred to as “PVdF”) polymers.
Particularly, PVdF-based membranes are mainly used as membranes for Western blotting, because they have excellent protein-binding sensitivity, membrane strength and handling properties compared to nitrocellulose- or nylon-based membranes.
Western blotting is a technique used to detect specific proteins in a given sample of tissue homogenate or extract. It is a technique for detecting any specific protein from a mixture of various proteins and is a method of detecting the presence of a protein by causing an antigen-antibody reaction using an antibody against to the protein to be detected.
Western blotting is also used to separate native gels or denatured proteins by the sizes of the three-dimensional structures of polypeptides or proteins.
In Western blotting, proteins extracted from cells or tissues are mixed with a sample buffer and placed on a molecular sieve made of acrylamide, followed by electrophoresis. Then, the sodium dodecylsulfate (SDS) contained in the sample buffer causes the proteins to carry negative (−) charges, such that the proteins are attracted toward positive (+) charges. At this time, the SDS molecular sieve interferes with the movement of the proteins, so that small molecules move fast, and large molecules move slowly, thus forming bands of various sizes. When a membrane is placed on the gels separated according to size and electricity is applied thereto, the proteins are transferred to the membrane. An antibody against a specific protein to be detected is bound to the membrane, a secondary antibody specific to the antibody is then bound to the membrane, and the resulting color development reactions are imaged by X-rays.
The membrane that is used for this purpose is a porous membrane which is made of a polymer (e.g., PVdF) capable of hydrophobic interaction with protein and has an average pore diameter of 0.2-0.45 μm.
This porous membrane is manufactured by a method such as wet, dry or dry-wet casting by phase separation, wherein a solvent and a polymer are introduced into a nonsolvent such as water. However, the porous membrane is manufactured at a high cost and difficult to manufacture in a large amount.
Also, if the porous membrane is manufactured by phase separation, there is a disadvantage in that the distribution of pore structures is not uniform. For example, a PVdF membrane needs to be subjected to a process of partially hydrophilizing the membrane by immersing it in methanol before use, thereby maximizing the compatibility of the membrane with buffer solution. If this hydrophilizing process is not performed, the sensitivity of the membrane can be reduced, because proteins are not sufficiently adsorbed on the membrane.
This methanol pretreatment process can greatly reduce the strength of the membrane to cause cracks and forms air bubbles to cause a background, thus making it difficult to precisely detect a desired protein.
Accordingly, the present inventors have used an electrospinning process to manufacture a nanofiber membrane which is expensive, manufactured in a simple and convenient manner, has an artificially controllable pore structure, is made of nanofibers having maximized surface area, and has excellent sensitivity compared to existing membranes even when it is not subjected to a methanol pretreatment process, thereby completing the present invention.